Minireview Emerging nanotechnologies for cancer immunotherapy Sourabh Shukla1,2 and Nicole F Steinmetz1,2,3,4,5 1
Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH 44106, USA; 2Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44106, USA; 3Department of Radiology, Case Western Reserve University, Cleveland, OH 44106, USA; 4Department of Materials Science and Engineering, Case Western Reserve University, Cleveland, OH 44106, USA; 5Department of Macromolecular Science and Engineering, Case Western Reserve University, Cleveland, OH 44106 Corresponding authors: Sourabh Shukla. Email: [email protected]; Nicole F Steinmetz. Email: [email protected]
Abstract Founded on the growing insight into the complex cancer-immune system interactions, adjuvant immunotherapies are rapidly emerging and being adapted for the treatment of various human malignancies. Immune checkpoint inhibitors, for example, have already shown clinical success. Nevertheless, many approaches are not optimized, require frequent administration, are associated with systemic toxicities and only show modest efficacy as monotherapies. Nanotechnology can potentially enhance the efficacy of such immunotherapies by improving the delivery, retention and release of immunostimulatory agents and biologicals in targeted cell populations and tissues. This review presents the current status and emerging trends in such nanotechnology-based cancer immunotherapies including the role of nanoparticles as carriers of immunomodulators, nanoparticles-based cancer vaccines, and depots for sustained immunostimulation. Also highlighted are key translational challenges and opportunities in this rapidly growing field. Keywords: Cancer immunotherapy, nanotechnology, vaccines, immunomodulation Experimental Biology and Medicine 2016; 241: 1116–1126. DOI: 10.1177/1535370216647123
Introduction Cancer immunotherapies – revitalizing the immune system against cancer One of the major developments in the fight against cancer is the emergence of immunotherapies that are aimed at harnessing the exquisite and specific power of the immune system against malignancies.1,2 The renewed excitement and push for novel immunotherapies stems from the success of two different strategies – adaptive T-cell therapy based on chimeric antigen receptors (CARs) and checkpoint blockade. The former, CAR T cells therapy, still under clinical evaluation, is based on genetically engineering patient’s own T-cells with CARs that recognizes tumor antigens. These CAR T cells are then expanded in vitro and infused back in patients, where they are likely to recognize and kill cancer cells.3 Checkpoint blockade therapies, on the other hand work by inhibiting pathways that keep the duration and strength of immune system in check.4 The recent approval of two checkpoint blockade therapies targeting the receptors CTLA-4 and PD-1 have come on the back of several successful clinical trials where treatment with checkpoint blockade inhibitors has resulted in striking T cell function restoration in melanoma, renal cell carcinoma and lung cancer.4,5 ISSN: 1535-3702 Copyright ß 2016 by the Society for Experimental Biology and Medicine
These developments and other similar efforts are clearly fueled by growing insights into the nature and consequences of interactions between tumors and the immune system, which frequently impede the development and function of anti-tumor immune response.6–8 The challenge for immunotherapies is to modulate such interactions towards successful recognition and elimination of cancer cells. The durability and specificity of such strategies has the potential to generate long-lived therapeutic effects with limited systemic toxicities.9,10 However, growing evidences suggest that immunotherapies could be most beneficial as an adjuvant therapy to conventional chemo- and radiation therapies.11 The premise of an immune system-mediated intervention in cancer progression lies in the capacity of the immune system to distinguish between self and nonself.12,13 While highly equipped and effective in the eradication of pathogens, the ability of the immune system to effectively deal with transforming cancer cells is hampered by the fact that the cancer cell’s origin is self, and because of depletion of self-antigen reactive T lymphocytes during development.14 Nevertheless, cancer is characterized by the accumulation of a variable number of genetic alterations and the loss of normal cellular regulatory processes, resulting in expression of neo-antigens arising from mutated Experimental Biology and Medicine 2016; 241: 1116–1126
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.......................................................................................................................... genes, chromosomal aberrations or overexpression of embryonic antigens.15,16 These neo-antigens differentiate cancer from normal cells and enable their immune-recognition, as indicated by the presence of basal levels of tumor antigen-specific cellular and humoral responses in subsets of cancer patients.13,17 However, a highly immunosuppressive microenvironment under the influence of cytokines such as TGF- and IL-2,18 along with immunosuppressive cells such as Foxp3þ Tregs (regulatory T cells),19 myeloidderived suppressor cells (MDSCs)20 and M2-type macrophages,21 or combinations thereof, keeps such autologous immune response in check. Moreover, through intricate immunoediting mechanisms, including antigen shedding, negative selection of antigenic cancer cells, down-regulation of MHC-I molecules and turning off activated T cells via negative regulators such as PD-1,4 cancer cells evade immunosurveillance and the tumor prevails.7 To overcome these hurdles and tip the scale in favor of anti-tumor immune response, a diverse set of immunotherapeutic approaches have been explored. While adaptive T cell transfer (ACT)22 and cancer vaccines23 are aimed at boosting tumor reactive immune cell populations, cytokines, immunomodulatory antibodies and small molecule drugs have been employed to overcome the immunosuppressive tumor microenvironment.24 The goal of cancer immunotherapy is to initiate or reinitiate a self-sustaining cycle of cancer immunity, enabling it to amplify and propagate.2 Although numerous such strategies have been explored, only a handful of them have been approved and adapted for clinical use with only a modest rate of success.1 Thus, there is a huge scope for further development in terms of specificity, enhanced effectiveness and reduced toxicities. Many immunotherapeutic strategies currently in preclinical or clinical evaluation are based on traditional drug development approaches where nanomedicine has already made significant contributions by improving stability, biodistribution through targeted delivery, bioavailability and efficacy of cytotoxic drugs or imaging contrast agents.25,26 Ongoing clinical evaluations of several candidate nanoformulations in conjugation with a wide range of chemotherapeutic or immunotherapeutic payloads are a testament of the efficacy of nanoparticle-based drug delivery approaches.27,28 For example, Nanoplatin NC-6004, a cisplatin containing polymeric nanocarrier is under Phase I/ II clinical trial,29 whereas cyclodextrin-based nanoparticles CALAA-01 that delivers small-interfering RNA (siRNA) agent to shut down a key enzyme (ribonucleotide reductase) in cancer cells is under Phase I evaluation.30 Similarly, an engineered adenovirus nanoparticle-based drug delivery platform is under Phase-1 dose escalation study for delivery of cancer immunotherapy to patients with chronic lymphocytic leukemia (CLL)31 while the pH sensitive polymeric nanoparticle CRLX101 loaded with camptothecin is undergoing Phase II clinical trials.32 Based on those very possibilities, an excellent opportunity for nanotechnology-mediated refinement exists in the field of immunotherapy.33–35 This review details some of the key immunotherapeutic strategies and highlights nanotechnology-based interventions that are being pursued to improve
the overall efficacy of such approaches, as summarized in Figure 1. Additionally, critical barriers to the successful translation of these emerging technologies are also discussed. Nanocarriers to deliver tumor microenvironment immunomodulators A number of immunomodulatory and immunostimulatory molecules such as cytokines, chemokines and targeted antibodies have been identified for their important roles in countering the highly immunosuppressive tumor microenvironment. Cytokine IL-2 promotes proliferation of effector functions of cytotoxic T lymphocytes (CTLs) and has shown clinical efficacy in malignant melanoma and renal carcinoma.36 IL-2 has also resulted in enhanced efficiency of other immunotherapies.37 Other cytokines such as IL-21 and IL-18 modulate both innate and adaptive immune responses through activation of CD4þ/CD8þ Tcells, natural killer (NK) cells, and B cells while suppressing Treg cells.38,39 Systemic administration of IL-21 and IL-18 also leads to enhanced production of IFN-, IL-2, tumor necrosis factor- (TNF-), granulocyte macrophage colony-stimulating factor (GM-CSF), IL-1 and IL-6 by activating T cells. Similarly, type I interferons (IFN- and ) demonstrate antitumor activities through stimulation of NK cell activity and suppression of allospecific suppressor T cells. Indeed, the administration of type I interferons has shown promise and efficacy in clinical trials in the setting of leukemia, melanoma and renal cell carcinoma.40 Type II interferons (IFN-) induce apoptosis, upregulate HLA-I and HLA-II and therefore promote antigen presentation in cancer cells, which in turn mediates tumor rejection and has shown efficacy in the setting of ACT therapies.41 Other non-specific immunomodulators such as Toll-like receptor (TLR7/9) agonists (e.g., synthetic oligonucleotides CpG) promote Th1 polarization, trigger activation of innate and adaptive immune responses, lead to dendritic cell (DC) activation and proliferation of CD4þ/CD8þ T cells and modulate suppressive functions of Treg cells.42,43 Even though the immunomodulatory effects of these small molecules in overturning the suppressive tumor microenvironment are well documented, drawbacks are associated with such therapies: Besides the short half-life, stability and bioavailability challenges akin to many conventional therapeutic candidates, systemic toxicities of cytokines arising due to their broad spectrum of biological activity on a wide variety of cells are major safety concerns.44–46 Cytokines could lead to non-specific lymphocyte activation in circulation and increased incidences of autoimmune and allergic responses. IL-1, IL-2, IL-6, TNF, and TGF-b could lead to modulation of hepatic metabolisms.9 Similarly, systemic administration of CD-40 agonist used to trigger CD40 signaling for activation of antigen presenting cells can lead to widespread symptoms of cytokine release syndrome, ocular inflammation, elevated levels of hepatic enzymes, and hematologic toxicities including T-cell depletion. Besides, agonistic anti-CD40 therapy has also been linked to long-term immunosuppression mediated by activation-induced apoptosis of CD4þ and CD8þ T cells.47,48 Similarly, overexposure to CpG could result in suppression
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.......................................................................................................................... of adaptive T cell immunity.49,50 Likewise, IL-2 administration at high doses causes vascular leak syndrome (VLS; also known as capillary leak syndrome), which is associated with increased vascular permeability, hypotension, pulmonary edema, liver cell damage and renal failure.51 Other side effects of IL-2 are hypothyroidism, thrombocytopenia, anemia, coagulopathy, or impairment of neutrophil chemotaxis, autoimmunity, neurotoxicity and myocarditis.52,53 In addition, the ability of IL-2 to stimulate TReg cells diminishes the beneficial effects of stimulating tumor-specific T cell response.54 Systemic administration of IL-2 with adaptively transferred T cells could also cause multi organ failures in severe cases. At the same time, dose dependent toxicities including thrombocytopenia, fatigue, and pyrexia have been associated with checkpoint blockade inhibitors.9 To overcome these challenges and provide a pathway for safe clinical use of immunomodulatory cytokines/chemokines, nanotechnology approaches hold great promise for the path forward. Nanoformulations consisting of such immunomodulatory molecules have improved bioavailability due to significantly prolonged circulation times of the carrier particles and in vivo stability of payload against serum inactivation and enzyme degradation.55,56 For example, intravenous administrations of liposomes containing cytokines such as IFN-g, IFN-a, IL-2 or TNF-a enhance the plasma residence time.55,57,58 On the other hand, intraperitoneal, intramuscular, subcutaneous or intranasal administration of cytokine carrying liposomes and polymeric particles creates local depots and increases residence times of the immunostimulatory payloads at the site (discussed in details later).59–61 Additionally, requirement of external or physiological stimuli ensures the release of immunostimulatory cargo only at targeted sites and further improve its bioavailability and safety.62–65 Specifically, nanoparticle-based delivery promotes the preferential accumulation and retention of immunomodulators in tumors due to the enhanced permeability and retention (EPR) effect, while minimizing off-target systemic toxicities, thereby improving potential for clinical translation of such therapies.66,67 Building on EPR effect-mediated nanoparticle homing, nanotechnologies are undergoing development targeting and modulating the immunosuppressive tumor microenvironment to attain efficacy of immunotherapies. For example, based on the passive tumor homing properties, lipid-coated calcium phosphate nanoparticles (LCP-NPs) have been used for tumor microenvironment immunomodulation by delivering TGF- siRNA and thereby down-regulating the levels immunosuppressive TGF- within the tumor. LCP-NPs have also been used to deliver a broad spectrum anti-inflammatory triterpenoid – methyl-2-cyano-3,12-dioxooleana-1,9(11)dien-28-oate (CDDO-Me) – that significantly reduced Treg and MDSC populations. The delivery of immunostimulatory molecules by LCP-NP has been combined with vaccination strategies using an LCP vaccine delivering a tumor antigen (Trp 2 peptide) and adjuvant (CpG oligonucleotide) to DCs – the combination therapy resulted in improved efficacy over vaccine only treatments.68,69
Similarly, EPR-mediated accumulation of liposomeencapsulated polymer nanogels has been utilized for intratumoral delivery of cytokine (IL-2) and TGF-b receptor I inhibitor—SB505124, a hydrophobic small molecule drug, leading to inhibition of TGF- receptor I and subsequent expansion of T cells and NK cells by blocking key immunosuppressive pathways.70 Similarly, by delivering PD-L1 siRNA using polyethylenimine (PEI) liposomes, PD-L1 levels have been knocked down leading to immunosuppressive to immunostimulatory phenotype changes in human and mouse ovarian cancer-associated DCs with subsequent increase in tumor-reactive CD8þ T cell numbers and improved mice survival.71 Liposomal delivery of IL-2 has also showed enhanced therapeutic effects with reduced toxicities in a variety of other tumors including liver and lung cancers leading to significant reduction in tumor growth.72,73 In addition to their use for systemic delivery and homing to tumors, nanoparticle formulations show benefits for intratumoral delivery of immunomodulators: based on their size, the nanoparticle-based formulation of immunotherapies limits their escape into systemic circulation, thus minimizing off-target effects and maximizing local immunostimulation. For example, immunostimulatory liposomes conjugated with IL-2 and anti-CD137 antibodies targeting activated T cells led to increased IL-2 dosing within the tumor when delivered directly via intratumoral vs. systemic injections. The intratumoral treatment resulted in a higher ratio of tumor-infiltrating CD8þ T cells over regulatory T cells in established melanomas.46 Likewise, PEGylated liposome formulation have been used to deliver agonistic anti-CD40 antibodies and TLR agonist CpG molecules using intratumoral administration resulting in significant tumor inhibition while sequestering the immunostimulatory payload in targeted tissues and reducing its systemic leakage, thus minimizing off-target inflammatory effects.45 Similarly, intratumoral administration of CpG payloads on gold nanoparticles has shown to induce significant macrophage and DC infiltration in tumors and significantly affected tumor growth by concentrating the CpG oligonucleotides in the tumor tissue and lowering the high dose requirements of systemic administrations.74 Passive tumor homing and intratumoral administration of nanoparticles are accompanied by their natural tropism towards phagocytic cells of the innate immune system in vivo including monocytes, neutrophils, macrophages and dendritic cells. Such affinity towards immune cells can also be used to deliver immunomodulating payloads to tumor-infiltrating immune cell populations and thereby reprogramming the tumor microenvironment. For example, nanocomplexes encapsulating CpG oligonucleotide and anti-IL-10 and anti-IL-10 receptor antisense oligonucleotides were efficiently captured by tumor-associated macrophages (TAMs) and resulted in altered macrophage phenotypes, leading to a significant anti-tumor effect in a hepatoma murine model.75 To improve the partition in TAMs over macrophages associated with the mononuclear phagocyte system (MPS), mannose-modified polymeric micelles containing acid-sensitive PEG modifications were developed. The PEG shielding reduced uptake by the MPS
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.......................................................................................................................... in circulation at neutral pH and improved tumor accumulation through persistent circulation. The acidic pH in the tumor resulted in PEG shedding and uptake by TAMs and their subsequent reprogramming.76 However, nanoparticles have also been targeted to immune cells in circulation to deliver payloads to the tumor tissue. RGD-targeted single walled carbon nanotubes (SWCNTs) have showed enhanced tumor accumulation via hitchhiking Ly6Chi monocytes in the circulation that are recruited to the site of the tumor in response to inflammation.77 Also, in a recent study, gold nanoparticles combining mouse vascular endothelial growth factor (VEGF)-siRNA with TAM-targeting M2 peptide have been used to inhibit both TAMs and cancer cells by targeting the VEGF pathway in both cell populations. Such synergistic inhibitory effects on both cancer cells and the immunosuppressive TAM population resulted in significant tumor regression and disease control for extended periods in orthotopic lung cancer in mice.78 These examples highlight the utility of nanoparticles to target phagocytic cells for immune activation; tissue- and cell-specificity can be further tailored by altering nanoparticle shape, size, charge, hydrophobicity and surface chemistry.33,79,80 In addition to their natural interactions with mononuclear phagocyte cells, nanoparticles carrying immunostimulatory payloads have also been utilized to modulate the functioning of T and B cells for therapeutic interventions. Adaptive T cell immunotherapy plays a central role in cancer therapy; cancer antigen-specific T cells can be expanded using vaccines (in vivo) or through ACT, the latter requires the ex vivo expansion of T cells followed by infusion into patient. While effective, ACT has many drawbacks because the methods are expensive, cumbersome and personalized.22 To enhance the technology, in one study, dextran-coated iron oxide particles with surface-coupled MHC-Ig dimers and anti-CD28 antibodies were designed to allow magnetic field-based aggregation of particles bound to T cell receptors (TCRs). Ex vivo stimulation of T cells with these particles in the presence of a magnetic fieldenhanced TCR clustering reduced the threshold of activation of T cells and improved the efficacy of adaptive T cell therapy.81 Moreover, nanotechnology opens the door for the in vivo targeting, priming and expansion of T cells. For example, in vivo loading of T cells with lipid nanoparticle ‘‘backpacks’’ carrying stimulatory cytokines was demonstrated. The nanoparticle-mediated in vivo priming resulted in 80-fold increased T cell expansion and significant enhancements in the efficacy of ACT without systemic toxicity.82 Similarly, circulating adaptive T cells were targeted in vivo by IL-2 loaded liposomes via anti-Thy1 antibodies, resulting in enhanced T cell proliferation more effectively compared to administration of soluble cytokines.83 These approaches overcome a decline in function of transplanted T cells following infusion, particularly in the setting of solid cancers with a highly immunosuppressive microenvironment. Finally, we recently demonstrated the use of virus-based nanoparticles as an immunotherapeutic, where the properties of the nanoparticle itself unlocked a potent anti-tumor immune response. We have demonstrated that plant-
derived virus-like particles stimulate a potent immunemediated anti-tumor response when introduced into the tumor microenvironment after tumors are established: VLPs from cowpea mosaic virus (CPMV), without nucleic acids, LPS, or any other recognized immune adjuvants, generated an effective anti-tumor immune response in mouse models of multiple tumor types, including triple negative breast cancer, disseminated ovarian cancer and primary and metastatic melanoma. The particles are not cytolytic to tumor cells and the effects are immune mediated. Most importantly, preliminary data indicate that the effect is systemic and durable, resulting in immune memory protecting mice from re-challenge.84 The immunotherapy follows an in situ vaccination approach in which immune-stimulatory reagents (here CPMV) are applied directly into the suspected metastatic site or into an identified primary tumor. This approach modulates the local microenvironment to relieve immunosuppression and potentiate anti-tumor immunity against antigens expressed by the tumor. This approach not only offers the potential for new therapeutics but also may lead to new levels of understanding how the immune system defines ‘‘danger signals’’. Improving cancer vaccines – mediators of adaptive immune response A wide range of cancer vaccines has been evaluated for a variety of human malignancies.85,86 The overarching goal is to deliver tumor-associated antigens to professional antigen presenting cells (APCs) to elicit adaptive immune responses mediated by tumor-specific cytotoxic T cells and antibodies. As an active immunotherapeutic approach aimed at stimulating endogenous anti-tumor immune response, cancer vaccines offer effective long-term protection against recurring and residual tumors. However, development of an effective therapeutic vaccine against established disease is challenging, and despite decades of pursuit, establishment of successful vaccination strategies based on proteins, peptides, autologous dendritic cells or tumor cells have largely been unsuccessful.18,87 While vaccines based on autologous cells are costly and technically challenging, peptide-based cancer vaccination suffers from inefficient uptake, processing and presentation of the delivered epitopes by activated professional APCs.23,51,87–90 Moreover, such vaccination strategies have led to the generation of low avidity tumorspecific T cell responses. Whole protein vaccination with powerful and often poorly tolerated adjuvants, immunostimulatory cytokines such as IL-2 or GM-CSF and/or TLR agonists have failed to induce clinically significant antitumor responses.91–93 However, spontaneous tumor antigen-specific and high avidity T cell response has been shown to control tumor progression. Together with the demonstration of significantly reduced tumor burden in both patients and animal models following ACT, this suggests that high avidity tumor-specific CTL response could lead to long-lasting immunoprotection against relapsing or residual cancer.85,86,94 Nanoparticle-based vaccine approaches offer multiple advantages that could fulfill the stringent requirements for the generation of such high avidity tumor-reactive
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.......................................................................................................................... T cells.95,96 Presenting antigens/epitopes on nanoparticulate carriers not only provides the requisite stability and longevity, it also facilitates efficient interactions with key immune cell populations.88,97 Clearly, nanoparticle engineering principles for vaccine platforms are different from those targeted for delivery of immunomodulators. While the former seeks interaction with APCs and other phagocytes, the latter aims to generally avoid phagocytic clearance thus prolonging circulation and improved tumor penetration. Nanoparticle-based vaccine formulations can improve the resulting immunostimulation by promoting multivalent receptor cross-linking, by altering intracellular processing and presentation or by colocalizing synergistic cues from the antigen, adjuvants and costimulatory molecules within the same cellular populations. The particulate nature of nanoparticles mimic pathogen-associated molecular patterns (PAMPs) that are perceived as danger signals and drive protective immunity.98 Such patterns are recognized by the pattern recognizing receptors (PRRs) such as TLRs on immune cells, specifically APCs, and facilitate enhanced uptake of nanoparticle-based vaccines by these cells.99,100 Activation of PPRs provides immunogenic cues to the immune system instructing it to launch specific response to the antigens carried by the nanoformulation. B cells have evolved to recognize multivalent display of antigens on microbial surfaces and play a major role in vaccine-mediated antibody responses, thus enhancement of their engagement is crucial for immunotherapies based on cancer vaccines. Nanoparticle-based antigen display that mimic pathogenic structural features are highly efficient in engaging B cell receptors (BCRs) to promote greater signaling, antigen internalization and processing of antigens for presentation to CD4þ T cells.101 Multivalent display of antigens on nanoparticles also leads to TLR stimulation promoting strong humoral responses with long-lived high avidity antibody responses mediated by secretion of co-stimulatory cytokines such as IFN- and IL-12.102 For example, we have demonstrated that potato virus X (PVX) displaying HER2-derived B-cell epitopes effectively generates HER2-specific antibodies.103 Similarly, plant viral nanoparticles coupled to a weak idiotypic (Id) tumor antigen have been used as a conjugate vaccine to induce antibody formation against a murine B-cell malignancy.104 Other virus-like particles have similarly been evaluated as efficient vaccine carriers.105–108 For a detailed review, we would like to refer the reader to our recent article (Lee, 2016).147. Professional APCs, particularly dendritic cells, are critical initiators of adaptive immune response, comprising both humoral and cellular responses, and are therefore an important target for anti-cancer nanomedicine. Nanoparticle-based vaccines are readily taken up by DCs and are associated with enhanced anti-tumor response as compared to soluble antigens.109 Following processing in endolysosomal compartments, soluble exogenous antigens are exclusively presented on MHC-II molecules to activate CD4þ helper T cells that in turn stimulate B cell-mediated antibody response. However, via a process called crosspresentation, nanoparticle-mediated delivery of antigenic peptides also results in cytosolic release of antigens where
MHC-I loading can occur, resulting in CD8þ T cell priming and ensuing cytotoxic T cell response.110 Such crosspresentation of cancer antigens have been demonstrated for a wide range of distinct nanoparticles and new strategies are being developed to improve the efficiency of such cross-presentation.111–113 These include strategies employed for cytosolic drug delivery of membrane-impermeable molecules such as via endolysosomal disruption through the proton sponge effect using biodegradable nanogels,114 endolysosomolytic and pH-responsive micelles,115 as well as endoplasmic reticulum (ER) targeting approaches where nanoparticles shuttle to cytosol following endosome-ER fusion.116 A significant advantage of nanoformulations is the control over their transport kinetics that facilitates tissue-specific delivery of antigens. Tumor antigens drain into tumor draining lymph nodes (TDLNs), where they are taken up by professional APCs, including DCs leading to subsequent presentation and priming of T cells. TDLNs are rich in phenotypically and functionally immature APCs and are therefore key targets for priming APCs using cancer vaccines and the subsequent adaptive immune response. The size-dependent lymphatic drainage of nanoformulations has been established and is an important design consideration for developing cancer vaccines: Nanoparticles between 20 and 45 nm optimally drain to lymph nodes and are retained.117 While smaller particles are likely to be flushed out of lymph nodes, those larger than 100 nm are less efficiently transported from the peripheral injection sites, generally via cell-mediated transport.118 Upon subcutaneous injection, only a small fraction of the vaccine is delivered to DCs whereas the majority is cleared by the body or engulfed by other immune cells. By targeting DCs, vaccine efficiencies can be further improved by overcoming non-specific uptake of nanoparticle-based vaccines. For example, cancer vaccines based on biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles when coated with an agonistic aCD40-mAb (NP-CD40), showed highly efficient and selective delivery to DCs in vivo and improved priming of CD8þ T cells against two independent tumor-associated antigens.119 Additionally, targeting a specific subset of DCs could also define the type of stimulated immune response.120 For example, TLR7 and TLR9 agonists can convert the tolerogenic plasmacytoid DCs to innate immunostimulatory types, whereas targeting various C-type lectins can modulate variable adaptive response. For instance, DC-SIGN, DEC-205, DNGR-1 and Langerin favors CD8þ T cell cellular (Th1) responses while CD4þ and B cell humoral (Th2) responses are achieved by targeting of DCIR2.121 The targeting of multiple DCs subtypes, in particular, holds great potential to enhance vaccine efficiencies. Sustained immunostimulation through in situ depots and artificial APCs Nanoparticles-based depots have recently gained attraction as a source of sustainable immune stimulus. For example, cytokine depots composed of liposomes or polymeric
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.......................................................................................................................... particles carrying pro-inflammatory cytokines have been developed for anticancer vaccines and for intratumoral administration for therapy. These cytokine-loaded particles can enhance tumor-specific immune response in conjugation with tumor antigen from irradiated tumor cells or coencapsulated with the cytokines.55 Using these strategies, GM-CSF encapsulated in polymer particles or IFN- g/IL-2 in liposomes caused increased leukocyte infiltration/ increased humoral response and enhanced cytolytic capacity of CD8þ T cells resulting in higher fraction of mice surviving melanoma challenge.122 Similar studies extended in human trials involved incorporation of tumor-specific idiotype isolated form follicular lymphoma patient into IL-2 containing liposomes and monthly vaccinations which resulted in sustained tumor-specific CD4þ and CD8þ T cell response and continuous remission.123 On the other hand, cytokine depots have been also employed to treat primary tumors with peri- or intratumoral injections. Here, primary tumors serve as the source of antigen while cytokine depot activate leukocytes in tumor microenvironment and promote immunotherapy against primary tumor and metastasized tumor cells. Local injection of polymeric microparticles loaded with IL-2 for the treatment of brain or liver tumors has showed that this approach was more effective at treating tumors and protecting against rechallenge than tumor cells engineered to express IL-2.124 Compared to treatment with soluble IL-2, liposomal IL-2 treatment of B16 melanoma bearing mice resulted in higher survival rates, slower tumor growth rates prior to resection and increased recruitment and protected mice against rechallenge with melanoma.124 Further studies using this strategy have involved particles with combinations of various cytokines including IL-12, TNF-a, GM-CSF or IL-18. Treatment of established tumors with IL-12 loaded particles prior to surgical resections promoted systemic anti-tumor immunity that prevented recurrence and metastasis.125 Furthermore, particles with combination of IL-12 and GM-CSF showed eradication of primary tumors through CD4þ and CD8þ cells, while the effect on metastasis was through NK/ NKT cells.126 The concept of immunotherapeutic depot has also been similarly used in therapeutic vaccine DepoVaxTM (DPX0907), which employs a liposome-based platform harboring custom formulated mixtures of CD8þ T-cell peptide epitopes, a tetanus toxoid derived Th epitope, and an adjuvant of choice (such as a toll-like receptor agonist) to provide signals for improved antigen presentation. The liposomes carry incorporated hydrophilic antigens and adjuvant directly into an oil medium such as Montanide ISA51 VG, entrapping all vaccine ingredients in a form suitable for efficient uptake, processing and presentation by antigenpresenting cells (APCs). Such DPX formulated vaccines have been shown to induce effective immune responses after a single-dose administration.127 Another development in this area is the design and engineering of artificial APCs (aAPCs) that are synthetic mimics of natural antigen presenting cells, to promote T cell activation and subsequent expansion, both ex vivo and in vivo. Essentially, aAPCs are particles to which proteins
required for T cell activation, such as MHC-epitope complexes, agonist anti-CD3 and agonist anti-CD28, have been conjugated.128 Both spatial and temporal organization of these signals during aAPC/T cell contact is important for efficient T cell activation. The first generation aAPCs were composed of solid, micron-sized polystyrene beads or with iron oxide cores and were used for ex vivo expansion of T cells. Their large size provided a large area of contact between aAPCs and T cells. However, the second generation of aAPCs engineered for in vivo applications were smaller particles at the nanometer size scale (
Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH 44106, USA; 2Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44106, USA; 3Department of Radiology, Case Western Reserve University, Cleveland, OH 44106, USA; 4Department of Materials Science and Engineering, Case Western Reserve University, Cleveland, OH 44106, USA; 5Department of Macromolecular Science and Engineering, Case Western Reserve University, Cleveland, OH 44106 Corresponding authors: Sourabh Shukla. Email: [email protected]; Nicole F Steinmetz. Email: [email protected]
Abstract Founded on the growing insight into the complex cancer-immune system interactions, adjuvant immunotherapies are rapidly emerging and being adapted for the treatment of various human malignancies. Immune checkpoint inhibitors, for example, have already shown clinical success. Nevertheless, many approaches are not optimized, require frequent administration, are associated with systemic toxicities and only show modest efficacy as monotherapies. Nanotechnology can potentially enhance the efficacy of such immunotherapies by improving the delivery, retention and release of immunostimulatory agents and biologicals in targeted cell populations and tissues. This review presents the current status and emerging trends in such nanotechnology-based cancer immunotherapies including the role of nanoparticles as carriers of immunomodulators, nanoparticles-based cancer vaccines, and depots for sustained immunostimulation. Also highlighted are key translational challenges and opportunities in this rapidly growing field. Keywords: Cancer immunotherapy, nanotechnology, vaccines, immunomodulation Experimental Biology and Medicine 2016; 241: 1116–1126. DOI: 10.1177/1535370216647123
Introduction Cancer immunotherapies – revitalizing the immune system against cancer One of the major developments in the fight against cancer is the emergence of immunotherapies that are aimed at harnessing the exquisite and specific power of the immune system against malignancies.1,2 The renewed excitement and push for novel immunotherapies stems from the success of two different strategies – adaptive T-cell therapy based on chimeric antigen receptors (CARs) and checkpoint blockade. The former, CAR T cells therapy, still under clinical evaluation, is based on genetically engineering patient’s own T-cells with CARs that recognizes tumor antigens. These CAR T cells are then expanded in vitro and infused back in patients, where they are likely to recognize and kill cancer cells.3 Checkpoint blockade therapies, on the other hand work by inhibiting pathways that keep the duration and strength of immune system in check.4 The recent approval of two checkpoint blockade therapies targeting the receptors CTLA-4 and PD-1 have come on the back of several successful clinical trials where treatment with checkpoint blockade inhibitors has resulted in striking T cell function restoration in melanoma, renal cell carcinoma and lung cancer.4,5 ISSN: 1535-3702 Copyright ß 2016 by the Society for Experimental Biology and Medicine
These developments and other similar efforts are clearly fueled by growing insights into the nature and consequences of interactions between tumors and the immune system, which frequently impede the development and function of anti-tumor immune response.6–8 The challenge for immunotherapies is to modulate such interactions towards successful recognition and elimination of cancer cells. The durability and specificity of such strategies has the potential to generate long-lived therapeutic effects with limited systemic toxicities.9,10 However, growing evidences suggest that immunotherapies could be most beneficial as an adjuvant therapy to conventional chemo- and radiation therapies.11 The premise of an immune system-mediated intervention in cancer progression lies in the capacity of the immune system to distinguish between self and nonself.12,13 While highly equipped and effective in the eradication of pathogens, the ability of the immune system to effectively deal with transforming cancer cells is hampered by the fact that the cancer cell’s origin is self, and because of depletion of self-antigen reactive T lymphocytes during development.14 Nevertheless, cancer is characterized by the accumulation of a variable number of genetic alterations and the loss of normal cellular regulatory processes, resulting in expression of neo-antigens arising from mutated Experimental Biology and Medicine 2016; 241: 1116–1126
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.......................................................................................................................... genes, chromosomal aberrations or overexpression of embryonic antigens.15,16 These neo-antigens differentiate cancer from normal cells and enable their immune-recognition, as indicated by the presence of basal levels of tumor antigen-specific cellular and humoral responses in subsets of cancer patients.13,17 However, a highly immunosuppressive microenvironment under the influence of cytokines such as TGF- and IL-2,18 along with immunosuppressive cells such as Foxp3þ Tregs (regulatory T cells),19 myeloidderived suppressor cells (MDSCs)20 and M2-type macrophages,21 or combinations thereof, keeps such autologous immune response in check. Moreover, through intricate immunoediting mechanisms, including antigen shedding, negative selection of antigenic cancer cells, down-regulation of MHC-I molecules and turning off activated T cells via negative regulators such as PD-1,4 cancer cells evade immunosurveillance and the tumor prevails.7 To overcome these hurdles and tip the scale in favor of anti-tumor immune response, a diverse set of immunotherapeutic approaches have been explored. While adaptive T cell transfer (ACT)22 and cancer vaccines23 are aimed at boosting tumor reactive immune cell populations, cytokines, immunomodulatory antibodies and small molecule drugs have been employed to overcome the immunosuppressive tumor microenvironment.24 The goal of cancer immunotherapy is to initiate or reinitiate a self-sustaining cycle of cancer immunity, enabling it to amplify and propagate.2 Although numerous such strategies have been explored, only a handful of them have been approved and adapted for clinical use with only a modest rate of success.1 Thus, there is a huge scope for further development in terms of specificity, enhanced effectiveness and reduced toxicities. Many immunotherapeutic strategies currently in preclinical or clinical evaluation are based on traditional drug development approaches where nanomedicine has already made significant contributions by improving stability, biodistribution through targeted delivery, bioavailability and efficacy of cytotoxic drugs or imaging contrast agents.25,26 Ongoing clinical evaluations of several candidate nanoformulations in conjugation with a wide range of chemotherapeutic or immunotherapeutic payloads are a testament of the efficacy of nanoparticle-based drug delivery approaches.27,28 For example, Nanoplatin NC-6004, a cisplatin containing polymeric nanocarrier is under Phase I/ II clinical trial,29 whereas cyclodextrin-based nanoparticles CALAA-01 that delivers small-interfering RNA (siRNA) agent to shut down a key enzyme (ribonucleotide reductase) in cancer cells is under Phase I evaluation.30 Similarly, an engineered adenovirus nanoparticle-based drug delivery platform is under Phase-1 dose escalation study for delivery of cancer immunotherapy to patients with chronic lymphocytic leukemia (CLL)31 while the pH sensitive polymeric nanoparticle CRLX101 loaded with camptothecin is undergoing Phase II clinical trials.32 Based on those very possibilities, an excellent opportunity for nanotechnology-mediated refinement exists in the field of immunotherapy.33–35 This review details some of the key immunotherapeutic strategies and highlights nanotechnology-based interventions that are being pursued to improve
the overall efficacy of such approaches, as summarized in Figure 1. Additionally, critical barriers to the successful translation of these emerging technologies are also discussed. Nanocarriers to deliver tumor microenvironment immunomodulators A number of immunomodulatory and immunostimulatory molecules such as cytokines, chemokines and targeted antibodies have been identified for their important roles in countering the highly immunosuppressive tumor microenvironment. Cytokine IL-2 promotes proliferation of effector functions of cytotoxic T lymphocytes (CTLs) and has shown clinical efficacy in malignant melanoma and renal carcinoma.36 IL-2 has also resulted in enhanced efficiency of other immunotherapies.37 Other cytokines such as IL-21 and IL-18 modulate both innate and adaptive immune responses through activation of CD4þ/CD8þ Tcells, natural killer (NK) cells, and B cells while suppressing Treg cells.38,39 Systemic administration of IL-21 and IL-18 also leads to enhanced production of IFN-, IL-2, tumor necrosis factor- (TNF-), granulocyte macrophage colony-stimulating factor (GM-CSF), IL-1 and IL-6 by activating T cells. Similarly, type I interferons (IFN- and ) demonstrate antitumor activities through stimulation of NK cell activity and suppression of allospecific suppressor T cells. Indeed, the administration of type I interferons has shown promise and efficacy in clinical trials in the setting of leukemia, melanoma and renal cell carcinoma.40 Type II interferons (IFN-) induce apoptosis, upregulate HLA-I and HLA-II and therefore promote antigen presentation in cancer cells, which in turn mediates tumor rejection and has shown efficacy in the setting of ACT therapies.41 Other non-specific immunomodulators such as Toll-like receptor (TLR7/9) agonists (e.g., synthetic oligonucleotides CpG) promote Th1 polarization, trigger activation of innate and adaptive immune responses, lead to dendritic cell (DC) activation and proliferation of CD4þ/CD8þ T cells and modulate suppressive functions of Treg cells.42,43 Even though the immunomodulatory effects of these small molecules in overturning the suppressive tumor microenvironment are well documented, drawbacks are associated with such therapies: Besides the short half-life, stability and bioavailability challenges akin to many conventional therapeutic candidates, systemic toxicities of cytokines arising due to their broad spectrum of biological activity on a wide variety of cells are major safety concerns.44–46 Cytokines could lead to non-specific lymphocyte activation in circulation and increased incidences of autoimmune and allergic responses. IL-1, IL-2, IL-6, TNF, and TGF-b could lead to modulation of hepatic metabolisms.9 Similarly, systemic administration of CD-40 agonist used to trigger CD40 signaling for activation of antigen presenting cells can lead to widespread symptoms of cytokine release syndrome, ocular inflammation, elevated levels of hepatic enzymes, and hematologic toxicities including T-cell depletion. Besides, agonistic anti-CD40 therapy has also been linked to long-term immunosuppression mediated by activation-induced apoptosis of CD4þ and CD8þ T cells.47,48 Similarly, overexposure to CpG could result in suppression
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.......................................................................................................................... of adaptive T cell immunity.49,50 Likewise, IL-2 administration at high doses causes vascular leak syndrome (VLS; also known as capillary leak syndrome), which is associated with increased vascular permeability, hypotension, pulmonary edema, liver cell damage and renal failure.51 Other side effects of IL-2 are hypothyroidism, thrombocytopenia, anemia, coagulopathy, or impairment of neutrophil chemotaxis, autoimmunity, neurotoxicity and myocarditis.52,53 In addition, the ability of IL-2 to stimulate TReg cells diminishes the beneficial effects of stimulating tumor-specific T cell response.54 Systemic administration of IL-2 with adaptively transferred T cells could also cause multi organ failures in severe cases. At the same time, dose dependent toxicities including thrombocytopenia, fatigue, and pyrexia have been associated with checkpoint blockade inhibitors.9 To overcome these challenges and provide a pathway for safe clinical use of immunomodulatory cytokines/chemokines, nanotechnology approaches hold great promise for the path forward. Nanoformulations consisting of such immunomodulatory molecules have improved bioavailability due to significantly prolonged circulation times of the carrier particles and in vivo stability of payload against serum inactivation and enzyme degradation.55,56 For example, intravenous administrations of liposomes containing cytokines such as IFN-g, IFN-a, IL-2 or TNF-a enhance the plasma residence time.55,57,58 On the other hand, intraperitoneal, intramuscular, subcutaneous or intranasal administration of cytokine carrying liposomes and polymeric particles creates local depots and increases residence times of the immunostimulatory payloads at the site (discussed in details later).59–61 Additionally, requirement of external or physiological stimuli ensures the release of immunostimulatory cargo only at targeted sites and further improve its bioavailability and safety.62–65 Specifically, nanoparticle-based delivery promotes the preferential accumulation and retention of immunomodulators in tumors due to the enhanced permeability and retention (EPR) effect, while minimizing off-target systemic toxicities, thereby improving potential for clinical translation of such therapies.66,67 Building on EPR effect-mediated nanoparticle homing, nanotechnologies are undergoing development targeting and modulating the immunosuppressive tumor microenvironment to attain efficacy of immunotherapies. For example, based on the passive tumor homing properties, lipid-coated calcium phosphate nanoparticles (LCP-NPs) have been used for tumor microenvironment immunomodulation by delivering TGF- siRNA and thereby down-regulating the levels immunosuppressive TGF- within the tumor. LCP-NPs have also been used to deliver a broad spectrum anti-inflammatory triterpenoid – methyl-2-cyano-3,12-dioxooleana-1,9(11)dien-28-oate (CDDO-Me) – that significantly reduced Treg and MDSC populations. The delivery of immunostimulatory molecules by LCP-NP has been combined with vaccination strategies using an LCP vaccine delivering a tumor antigen (Trp 2 peptide) and adjuvant (CpG oligonucleotide) to DCs – the combination therapy resulted in improved efficacy over vaccine only treatments.68,69
Similarly, EPR-mediated accumulation of liposomeencapsulated polymer nanogels has been utilized for intratumoral delivery of cytokine (IL-2) and TGF-b receptor I inhibitor—SB505124, a hydrophobic small molecule drug, leading to inhibition of TGF- receptor I and subsequent expansion of T cells and NK cells by blocking key immunosuppressive pathways.70 Similarly, by delivering PD-L1 siRNA using polyethylenimine (PEI) liposomes, PD-L1 levels have been knocked down leading to immunosuppressive to immunostimulatory phenotype changes in human and mouse ovarian cancer-associated DCs with subsequent increase in tumor-reactive CD8þ T cell numbers and improved mice survival.71 Liposomal delivery of IL-2 has also showed enhanced therapeutic effects with reduced toxicities in a variety of other tumors including liver and lung cancers leading to significant reduction in tumor growth.72,73 In addition to their use for systemic delivery and homing to tumors, nanoparticle formulations show benefits for intratumoral delivery of immunomodulators: based on their size, the nanoparticle-based formulation of immunotherapies limits their escape into systemic circulation, thus minimizing off-target effects and maximizing local immunostimulation. For example, immunostimulatory liposomes conjugated with IL-2 and anti-CD137 antibodies targeting activated T cells led to increased IL-2 dosing within the tumor when delivered directly via intratumoral vs. systemic injections. The intratumoral treatment resulted in a higher ratio of tumor-infiltrating CD8þ T cells over regulatory T cells in established melanomas.46 Likewise, PEGylated liposome formulation have been used to deliver agonistic anti-CD40 antibodies and TLR agonist CpG molecules using intratumoral administration resulting in significant tumor inhibition while sequestering the immunostimulatory payload in targeted tissues and reducing its systemic leakage, thus minimizing off-target inflammatory effects.45 Similarly, intratumoral administration of CpG payloads on gold nanoparticles has shown to induce significant macrophage and DC infiltration in tumors and significantly affected tumor growth by concentrating the CpG oligonucleotides in the tumor tissue and lowering the high dose requirements of systemic administrations.74 Passive tumor homing and intratumoral administration of nanoparticles are accompanied by their natural tropism towards phagocytic cells of the innate immune system in vivo including monocytes, neutrophils, macrophages and dendritic cells. Such affinity towards immune cells can also be used to deliver immunomodulating payloads to tumor-infiltrating immune cell populations and thereby reprogramming the tumor microenvironment. For example, nanocomplexes encapsulating CpG oligonucleotide and anti-IL-10 and anti-IL-10 receptor antisense oligonucleotides were efficiently captured by tumor-associated macrophages (TAMs) and resulted in altered macrophage phenotypes, leading to a significant anti-tumor effect in a hepatoma murine model.75 To improve the partition in TAMs over macrophages associated with the mononuclear phagocyte system (MPS), mannose-modified polymeric micelles containing acid-sensitive PEG modifications were developed. The PEG shielding reduced uptake by the MPS
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.......................................................................................................................... in circulation at neutral pH and improved tumor accumulation through persistent circulation. The acidic pH in the tumor resulted in PEG shedding and uptake by TAMs and their subsequent reprogramming.76 However, nanoparticles have also been targeted to immune cells in circulation to deliver payloads to the tumor tissue. RGD-targeted single walled carbon nanotubes (SWCNTs) have showed enhanced tumor accumulation via hitchhiking Ly6Chi monocytes in the circulation that are recruited to the site of the tumor in response to inflammation.77 Also, in a recent study, gold nanoparticles combining mouse vascular endothelial growth factor (VEGF)-siRNA with TAM-targeting M2 peptide have been used to inhibit both TAMs and cancer cells by targeting the VEGF pathway in both cell populations. Such synergistic inhibitory effects on both cancer cells and the immunosuppressive TAM population resulted in significant tumor regression and disease control for extended periods in orthotopic lung cancer in mice.78 These examples highlight the utility of nanoparticles to target phagocytic cells for immune activation; tissue- and cell-specificity can be further tailored by altering nanoparticle shape, size, charge, hydrophobicity and surface chemistry.33,79,80 In addition to their natural interactions with mononuclear phagocyte cells, nanoparticles carrying immunostimulatory payloads have also been utilized to modulate the functioning of T and B cells for therapeutic interventions. Adaptive T cell immunotherapy plays a central role in cancer therapy; cancer antigen-specific T cells can be expanded using vaccines (in vivo) or through ACT, the latter requires the ex vivo expansion of T cells followed by infusion into patient. While effective, ACT has many drawbacks because the methods are expensive, cumbersome and personalized.22 To enhance the technology, in one study, dextran-coated iron oxide particles with surface-coupled MHC-Ig dimers and anti-CD28 antibodies were designed to allow magnetic field-based aggregation of particles bound to T cell receptors (TCRs). Ex vivo stimulation of T cells with these particles in the presence of a magnetic fieldenhanced TCR clustering reduced the threshold of activation of T cells and improved the efficacy of adaptive T cell therapy.81 Moreover, nanotechnology opens the door for the in vivo targeting, priming and expansion of T cells. For example, in vivo loading of T cells with lipid nanoparticle ‘‘backpacks’’ carrying stimulatory cytokines was demonstrated. The nanoparticle-mediated in vivo priming resulted in 80-fold increased T cell expansion and significant enhancements in the efficacy of ACT without systemic toxicity.82 Similarly, circulating adaptive T cells were targeted in vivo by IL-2 loaded liposomes via anti-Thy1 antibodies, resulting in enhanced T cell proliferation more effectively compared to administration of soluble cytokines.83 These approaches overcome a decline in function of transplanted T cells following infusion, particularly in the setting of solid cancers with a highly immunosuppressive microenvironment. Finally, we recently demonstrated the use of virus-based nanoparticles as an immunotherapeutic, where the properties of the nanoparticle itself unlocked a potent anti-tumor immune response. We have demonstrated that plant-
derived virus-like particles stimulate a potent immunemediated anti-tumor response when introduced into the tumor microenvironment after tumors are established: VLPs from cowpea mosaic virus (CPMV), without nucleic acids, LPS, or any other recognized immune adjuvants, generated an effective anti-tumor immune response in mouse models of multiple tumor types, including triple negative breast cancer, disseminated ovarian cancer and primary and metastatic melanoma. The particles are not cytolytic to tumor cells and the effects are immune mediated. Most importantly, preliminary data indicate that the effect is systemic and durable, resulting in immune memory protecting mice from re-challenge.84 The immunotherapy follows an in situ vaccination approach in which immune-stimulatory reagents (here CPMV) are applied directly into the suspected metastatic site or into an identified primary tumor. This approach modulates the local microenvironment to relieve immunosuppression and potentiate anti-tumor immunity against antigens expressed by the tumor. This approach not only offers the potential for new therapeutics but also may lead to new levels of understanding how the immune system defines ‘‘danger signals’’. Improving cancer vaccines – mediators of adaptive immune response A wide range of cancer vaccines has been evaluated for a variety of human malignancies.85,86 The overarching goal is to deliver tumor-associated antigens to professional antigen presenting cells (APCs) to elicit adaptive immune responses mediated by tumor-specific cytotoxic T cells and antibodies. As an active immunotherapeutic approach aimed at stimulating endogenous anti-tumor immune response, cancer vaccines offer effective long-term protection against recurring and residual tumors. However, development of an effective therapeutic vaccine against established disease is challenging, and despite decades of pursuit, establishment of successful vaccination strategies based on proteins, peptides, autologous dendritic cells or tumor cells have largely been unsuccessful.18,87 While vaccines based on autologous cells are costly and technically challenging, peptide-based cancer vaccination suffers from inefficient uptake, processing and presentation of the delivered epitopes by activated professional APCs.23,51,87–90 Moreover, such vaccination strategies have led to the generation of low avidity tumorspecific T cell responses. Whole protein vaccination with powerful and often poorly tolerated adjuvants, immunostimulatory cytokines such as IL-2 or GM-CSF and/or TLR agonists have failed to induce clinically significant antitumor responses.91–93 However, spontaneous tumor antigen-specific and high avidity T cell response has been shown to control tumor progression. Together with the demonstration of significantly reduced tumor burden in both patients and animal models following ACT, this suggests that high avidity tumor-specific CTL response could lead to long-lasting immunoprotection against relapsing or residual cancer.85,86,94 Nanoparticle-based vaccine approaches offer multiple advantages that could fulfill the stringent requirements for the generation of such high avidity tumor-reactive
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.......................................................................................................................... T cells.95,96 Presenting antigens/epitopes on nanoparticulate carriers not only provides the requisite stability and longevity, it also facilitates efficient interactions with key immune cell populations.88,97 Clearly, nanoparticle engineering principles for vaccine platforms are different from those targeted for delivery of immunomodulators. While the former seeks interaction with APCs and other phagocytes, the latter aims to generally avoid phagocytic clearance thus prolonging circulation and improved tumor penetration. Nanoparticle-based vaccine formulations can improve the resulting immunostimulation by promoting multivalent receptor cross-linking, by altering intracellular processing and presentation or by colocalizing synergistic cues from the antigen, adjuvants and costimulatory molecules within the same cellular populations. The particulate nature of nanoparticles mimic pathogen-associated molecular patterns (PAMPs) that are perceived as danger signals and drive protective immunity.98 Such patterns are recognized by the pattern recognizing receptors (PRRs) such as TLRs on immune cells, specifically APCs, and facilitate enhanced uptake of nanoparticle-based vaccines by these cells.99,100 Activation of PPRs provides immunogenic cues to the immune system instructing it to launch specific response to the antigens carried by the nanoformulation. B cells have evolved to recognize multivalent display of antigens on microbial surfaces and play a major role in vaccine-mediated antibody responses, thus enhancement of their engagement is crucial for immunotherapies based on cancer vaccines. Nanoparticle-based antigen display that mimic pathogenic structural features are highly efficient in engaging B cell receptors (BCRs) to promote greater signaling, antigen internalization and processing of antigens for presentation to CD4þ T cells.101 Multivalent display of antigens on nanoparticles also leads to TLR stimulation promoting strong humoral responses with long-lived high avidity antibody responses mediated by secretion of co-stimulatory cytokines such as IFN- and IL-12.102 For example, we have demonstrated that potato virus X (PVX) displaying HER2-derived B-cell epitopes effectively generates HER2-specific antibodies.103 Similarly, plant viral nanoparticles coupled to a weak idiotypic (Id) tumor antigen have been used as a conjugate vaccine to induce antibody formation against a murine B-cell malignancy.104 Other virus-like particles have similarly been evaluated as efficient vaccine carriers.105–108 For a detailed review, we would like to refer the reader to our recent article (Lee, 2016).147. Professional APCs, particularly dendritic cells, are critical initiators of adaptive immune response, comprising both humoral and cellular responses, and are therefore an important target for anti-cancer nanomedicine. Nanoparticle-based vaccines are readily taken up by DCs and are associated with enhanced anti-tumor response as compared to soluble antigens.109 Following processing in endolysosomal compartments, soluble exogenous antigens are exclusively presented on MHC-II molecules to activate CD4þ helper T cells that in turn stimulate B cell-mediated antibody response. However, via a process called crosspresentation, nanoparticle-mediated delivery of antigenic peptides also results in cytosolic release of antigens where
MHC-I loading can occur, resulting in CD8þ T cell priming and ensuing cytotoxic T cell response.110 Such crosspresentation of cancer antigens have been demonstrated for a wide range of distinct nanoparticles and new strategies are being developed to improve the efficiency of such cross-presentation.111–113 These include strategies employed for cytosolic drug delivery of membrane-impermeable molecules such as via endolysosomal disruption through the proton sponge effect using biodegradable nanogels,114 endolysosomolytic and pH-responsive micelles,115 as well as endoplasmic reticulum (ER) targeting approaches where nanoparticles shuttle to cytosol following endosome-ER fusion.116 A significant advantage of nanoformulations is the control over their transport kinetics that facilitates tissue-specific delivery of antigens. Tumor antigens drain into tumor draining lymph nodes (TDLNs), where they are taken up by professional APCs, including DCs leading to subsequent presentation and priming of T cells. TDLNs are rich in phenotypically and functionally immature APCs and are therefore key targets for priming APCs using cancer vaccines and the subsequent adaptive immune response. The size-dependent lymphatic drainage of nanoformulations has been established and is an important design consideration for developing cancer vaccines: Nanoparticles between 20 and 45 nm optimally drain to lymph nodes and are retained.117 While smaller particles are likely to be flushed out of lymph nodes, those larger than 100 nm are less efficiently transported from the peripheral injection sites, generally via cell-mediated transport.118 Upon subcutaneous injection, only a small fraction of the vaccine is delivered to DCs whereas the majority is cleared by the body or engulfed by other immune cells. By targeting DCs, vaccine efficiencies can be further improved by overcoming non-specific uptake of nanoparticle-based vaccines. For example, cancer vaccines based on biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles when coated with an agonistic aCD40-mAb (NP-CD40), showed highly efficient and selective delivery to DCs in vivo and improved priming of CD8þ T cells against two independent tumor-associated antigens.119 Additionally, targeting a specific subset of DCs could also define the type of stimulated immune response.120 For example, TLR7 and TLR9 agonists can convert the tolerogenic plasmacytoid DCs to innate immunostimulatory types, whereas targeting various C-type lectins can modulate variable adaptive response. For instance, DC-SIGN, DEC-205, DNGR-1 and Langerin favors CD8þ T cell cellular (Th1) responses while CD4þ and B cell humoral (Th2) responses are achieved by targeting of DCIR2.121 The targeting of multiple DCs subtypes, in particular, holds great potential to enhance vaccine efficiencies. Sustained immunostimulation through in situ depots and artificial APCs Nanoparticles-based depots have recently gained attraction as a source of sustainable immune stimulus. For example, cytokine depots composed of liposomes or polymeric
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.......................................................................................................................... particles carrying pro-inflammatory cytokines have been developed for anticancer vaccines and for intratumoral administration for therapy. These cytokine-loaded particles can enhance tumor-specific immune response in conjugation with tumor antigen from irradiated tumor cells or coencapsulated with the cytokines.55 Using these strategies, GM-CSF encapsulated in polymer particles or IFN- g/IL-2 in liposomes caused increased leukocyte infiltration/ increased humoral response and enhanced cytolytic capacity of CD8þ T cells resulting in higher fraction of mice surviving melanoma challenge.122 Similar studies extended in human trials involved incorporation of tumor-specific idiotype isolated form follicular lymphoma patient into IL-2 containing liposomes and monthly vaccinations which resulted in sustained tumor-specific CD4þ and CD8þ T cell response and continuous remission.123 On the other hand, cytokine depots have been also employed to treat primary tumors with peri- or intratumoral injections. Here, primary tumors serve as the source of antigen while cytokine depot activate leukocytes in tumor microenvironment and promote immunotherapy against primary tumor and metastasized tumor cells. Local injection of polymeric microparticles loaded with IL-2 for the treatment of brain or liver tumors has showed that this approach was more effective at treating tumors and protecting against rechallenge than tumor cells engineered to express IL-2.124 Compared to treatment with soluble IL-2, liposomal IL-2 treatment of B16 melanoma bearing mice resulted in higher survival rates, slower tumor growth rates prior to resection and increased recruitment and protected mice against rechallenge with melanoma.124 Further studies using this strategy have involved particles with combinations of various cytokines including IL-12, TNF-a, GM-CSF or IL-18. Treatment of established tumors with IL-12 loaded particles prior to surgical resections promoted systemic anti-tumor immunity that prevented recurrence and metastasis.125 Furthermore, particles with combination of IL-12 and GM-CSF showed eradication of primary tumors through CD4þ and CD8þ cells, while the effect on metastasis was through NK/ NKT cells.126 The concept of immunotherapeutic depot has also been similarly used in therapeutic vaccine DepoVaxTM (DPX0907), which employs a liposome-based platform harboring custom formulated mixtures of CD8þ T-cell peptide epitopes, a tetanus toxoid derived Th epitope, and an adjuvant of choice (such as a toll-like receptor agonist) to provide signals for improved antigen presentation. The liposomes carry incorporated hydrophilic antigens and adjuvant directly into an oil medium such as Montanide ISA51 VG, entrapping all vaccine ingredients in a form suitable for efficient uptake, processing and presentation by antigenpresenting cells (APCs). Such DPX formulated vaccines have been shown to induce effective immune responses after a single-dose administration.127 Another development in this area is the design and engineering of artificial APCs (aAPCs) that are synthetic mimics of natural antigen presenting cells, to promote T cell activation and subsequent expansion, both ex vivo and in vivo. Essentially, aAPCs are particles to which proteins
required for T cell activation, such as MHC-epitope complexes, agonist anti-CD3 and agonist anti-CD28, have been conjugated.128 Both spatial and temporal organization of these signals during aAPC/T cell contact is important for efficient T cell activation. The first generation aAPCs were composed of solid, micron-sized polystyrene beads or with iron oxide cores and were used for ex vivo expansion of T cells. Their large size provided a large area of contact between aAPCs and T cells. However, the second generation of aAPCs engineered for in vivo applications were smaller particles at the nanometer size scale (
